Food Biotechnology

ISSN: 0890-5436 (Print) 1532-4249 (Online) Journal homepage: http://www.tandfonline.com/loi/lfbt20

Phenotypic and Genomic Characterization

of Enterococcus Species from Some Nigerian
Fermented Foods
Iyabo Christianah Oladipo , Abiodun Sanni & Snehasiktas Swarnakar
To cite this article: Iyabo Christianah Oladipo , Abiodun Sanni & Snehasiktas Swarnakar (2013)
Phenotypic and Genomic Characterization of Enterococcus Species from Some Nigerian
Fermented Foods, Food Biotechnology, 27:1, 39-53, DOI: 10.1080/08905436.2012.755627
To link to this article: http://dx.doi.org/10.1080/08905436.2012.755627

Published online: 08 Feb 2013.

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Date: 01 November 2016, At: 05:13

Food Biotechnology, 27:3953, 2013

Copyright Taylor & Francis Group, LLC
ISSN: 0890-5436 print / 1532-4249 online
DOI: 10.1080/08905436.2012.755627

Phenotypic and Genomic

Characterization of
Enterococcus Species from
Some Nigerian Fermented
Foods
Iyabo Christianah Oladipo1,2, Abiodun Sanni1,
and Snehasiktas Swarnakar3
1

University of Ibadan, Microbiology, Oyo State, Nigeria, Ibadan, Nigeria

Ladoke Akintola University of Technology, Science Laboratory Technology, Ogbomoso,
Nigeria
3
Indian Institute of Chemical Biology, Physiology, Jadavpur, Kolkata, India
2

The gram-positive Enterococci bacteria are generally used as a starter and probiotic cultures in foods. However, they have emerged as one of the leading causes of nosocomial
infections worldwide, and this feature is aggravated by the development of antibiotic
resistance. Accurate identification of Enterococci at the species level is an important
task in food microbiology. In this study, 144 strains of Enterococcus species were isolated from traditional fermented vegetable condiment and West African soft cheese
(wara) with the most predominant species being E. gallinarum (75%) followed by E. faecium (14.5%), E. faecalis (7.6%), and E. casselliflavus (2.8%). The strains isolated were
characterized and identified using the polyphasic taxonomy approach. Phenotypically,
108 strains were characterized and identified to be E. gallinarum, 21 strains as E. faecium, 11 strains as E. faecalis, and 4 strains as E. casselliflavus. Thirty representative
strains were also subjected to genomic characterization, and the result obtained with
the phenotypic approach was confirmed. Therefore, the polyphasic taxonomy approach
was successful in the accurate identification of the Enterococcus species isolated.
Key Words: Enterococcus species; phenotypic; genomic; characterization; condiments

INTRODUCTION
Enterococci are typical lactic acid bacteria (LAB) important in food and clinical microbiology. They are gram-positive, nonspore forming, catalase-negative,
Address correspondence to Dr. Iyabo Christianah Oladipo, Ladoke Akintola
University of Technology, Science Laboratory Technology, Ogbomoso, Nigeria; E-mail:
xtiecoker@gmail.com

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I. C. Oladipo et al.

oxidase-negative, and facultative anaerobic bacteria that occur singly, in pairs,

or in chains. Most strains of the species are harmless commensal and may
be beneficial for health (probiotic). Also, they can be found naturally in raw
material and can be used in starter cultures for fermented foods. Other strains
are feared opportunistic pathogens, causing serious illnesses in hospitals all
over the world (Aakra et al., 2005). Organisms of the genus Enterococcus, and
in particular Enterococcus faecalis, have become a significant cause of nosocomial infections and usually show multiple drug resistance (Murray, 2000).
Resistance to the most commonly used antibiotics for gram-positive bacteria
provides these organisms with a selective advantage in the hospital environment (Murray, 1990). According to many authors, Enterococcus faecalis has
been successfully used to accelerate maturation and to improve organoleptic characteristic of cheeses (Ledda et al., 1994; Villani and Coppola, 1994).
On the basis of well-documented desirable biochemical properties which augmented technological acceptability of Enterococci, they have been proposed as
a part of defined starter cultures for different European cheeses such as WaterBuffalo Mozarella (Villani and Coppola, 1994), Feta (Litopoulou-Tzanetaki
et al., 1993), Venaco (Casalta and Zennaro, 1997), and Cebreiro (Centeno et al.,
1996) cheese.
Moreover, the ability of Enterococci to produce antimicrobial peptides,
known as bacteriocins is established which could be used as food/feed biopreservatives.. Bacteriocins are small, ribosomally synthesized, extracellular
and heat-stable peptides that exert antimicrobial activity against grampositive and gram-negative bacteria, including food spoilage or pathogenic
bacteria such as Listeria monocytogenes, Staphylococcus aureus, Clostridium
spp., Bacillus spp., and Campylobacter spp. (Casaus et al., 1997; Cintas et al.,
2001; Moreno et al., 2006; Nes et al., 2007; Line et al., 2008; Poeta et al., 2008).
Furthermore, a strain of E. faecium SF68 has been confirmed as a probiotic
due to the positive effects the bacteria produced against diarrhea in humans
and pigs. Despite these benefits and risks, there is no consensus whether these
bacteria pose the risk in food fermentation process because of their ability
to develop resistance against most antibiotics currently used in combination
with known virulent factors. The strains of Enterococci are naturally tolerant
to many antibacterial drugs including -lactam, cephalosporins, licosamidis
and polymyxins. A specific cause for concern and a factor contributing to the
pathogenesis of Enterococci is the resistance they acquire to aminoglycosidase, tetracyclines, macrolidis, chloramphenicol, penicillin, and ampicillin and
their capacity to exchange genetic information by conjugation (Gray et al.,
1991).
The present study was undertaken with the aim of isolating and identifying the Enterococci species from traditionally fermented vegetable condiments and West African soft cheese using both phenotypic and molecular
methods.

Genomic Characterization of Enterococcus Species

MATERIALS AND METHODS

Sample Collection
Samples of West African soft cheese (wara) and traditionally fermented
vegetable condiments (ugba, ogiri, okpehe, iru, and dawadawa) weighing
between 100 g and 150 g were randomly purchased from local markets
in Nigeria. The samples were separately packaged in polythene bags and
aseptically transported to the laboratory under cold conditions for analysis.

Microbiological Analysis
For isolation of presumptive Enterococci, 10 g of each sample were separately homogenized in 90 mL of sterile peptone water. Serial 10-fold dilutions
were performed and aliquots were plated on Slanetz and Bartley Medium
(Oxoid, Canada). After 48 h incubation at 37 C, typical small pinkish colonies of
presumptive Enterococci were randomly picked from plates and subcultured to
obtain pure isolates. Presumptive Enterococci isolates were cultured in Brain
Heart Infusion broth (Oxoid, Canada) with incubation at 37 C for 18 h. Pure
cultures were kept frozen at 20 C in BHI broth containing glycerol (50%).

Phenotypic Identification
Isolates were subjected to standard cultural, morphological and physiological techniques and identification according to Schleifer and Kilpper-Balz
(1984). After gram staining and catalase test, strains were preliminarily identified based on phenotypic properties such as ammonia (NH3 ) production from
arginine, ability to hydrolyse esculin and pyrrolidonyl--naphthylamide, ability to grow at 6.5% sodium chloride (NaCl), growth at pH 9.6, and ability to
grow at 10 C and 45 C. All the Enterococci strains were tested for the sugar
fermentation patterns and were differentiated on this basis.

Genomic Characterization
Isolation of Chromosomal DNA
To characterize the strains isolated using genomic tools, 30 representative
isolates were selected out of the 144 strains based on their ability to survive low
pH, growth at 6.5% sodium chloride (NaCl) concentration and ability to grow at
different temperature ranges. DNA was isolated from the 30 strains using the
modified method of Sambrook et al. (1989). Briefly, a single colony of bacteria
was inoculated in BHI broth and grown overnight. Cells were harvested from
5 mL of culture by centrifuging at 6000 rpm for 8 min at 4 C. The cells were
washed in 2 changes of 2 mL STE buffer (0.1M NaCl, 10 mM Tris pH 8 1 mM

41

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I. C. Oladipo et al.

EDTA pH 8), then 2 mL homogenization buffer (0.1 M NaCl, 0.3 M Tris pH 8,

0.2 M Sucrose, 0.1 M EDTA) was added to the pellet and mixed together by
pipetting up and down, 300 L of 10% SDS and 3 L of RNase A was added,
this was incubated at 65 C for 3 h with vortexing every 30 min. After incubation, the mixture of phenol: chloroform: isoamyl alcohol (25:24:1) was added
and centrifuged at 10,000 rpm for 10 min at 4 C. The aqueous phase was collected and the organic phase discarded; the DNA in the aqueous phase was
ethanol precipitated and kept overnight at 20 C then washed in 70% alcohol,
air dried, and dissolved in 50 L of Tris buffer. The quality of the DNA was
checked by running on 0.7% agarose gel stained with ethidium bromide 0.5
g/mL. A single intense band without smearing was noted. The extracted DNA
of the bacteria was used as template DNA for amplification of 16S rRNA gene.
Amplification of 16Sr RNA gene
Individual reactions (25 L) contained 50 ng of template DNA along
with master mix that included 1X PCR buffer, 10 mM dNTPs, 25 mM
MgCl2 , 1 unit of Taq DNA polymerase (Platinum Taq polymerase, Invitrogen,
Carlsbad, Calif., USA), and 100 pmol of each oligonucleotide primers designated as FD1 (5-AGAGTT TGATCCTGGCTCAG - 3) for ward and RD1 (5AAGGAGGTGATCCAGCC- 3) for reverse (Weisburg et al., 1991). The region
of 16S rRNA gene was amplified by initial denaturation for 3 min at 96 C, followed by 5 cycles of 1 min at 95 C, annealing at 50 C for 30 s, extension at 72 C
for 2 min and 30 cycles of 95 C for 1 min, annealing at 52 C for 30 s, extension
at 72 C for 2 min, and a final extension of 72 C for 7 min.
The amplified 1500-bp fragments were resolved by electrophoresis on a 1%
agarose gel stained with ethidium bromide and visualized under UV transilluminator. Subsequently, PCR products having the predicted sizes were
recovered from the gel with a QIAquick gel extraction kit (QIAGEN Inc.,
Canada).
Cloning
Cloning was done by using InsTAcloneTM PCR Cloning kit (Fermentas).
Ligation: Ligation reaction mixture included vector pTZ57R/T (0.18 pmol
ends), 5X ligation buffer, PCR product (0.54 pmol ends) and T4 DNA ligase.
This ligation mixture was spun down and incubated at 4 C overnight. During
ligation competent E. coli cells were prepared by transferring a single colony of
E. coli JM107 into 2 mL of C-medium, and the culture was incubated overnight
at 37 C in shaker.
Transformation: A 15 L volume of E. coli culture was introduced to 1.5 ml
of prewarmed C-medium and incubated at 37 C for 3 h, which was centrifuged
at 4500 rpm for 5 min and the supernatant discarded. Then the pellet was
resuspended in 300 L of solution-T and incubated on ice for 5 min. This was

Genomic Characterization of Enterococcus Species

centrifuged at 4500 rpm for 4 min and the supernatant discarded. Again the
pellet was resuspended in 120 L of solution-T and incubated on ice for 5 min.
After, 60 L of this was introduced to 10 L of ligation mixture and incubated on ice for 30 min. Whole mixture was immediately plated on prewarmed
LB-ampicillin-X Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside)/IPTG
(Isopropyl--D-thiogalacto-pyranoside) agar plates. The plates were incubated
overnight at 37 C, after which the plates were placed at 4 C for 5 h. The plates
were then warmed at 37 C for 20 min. Resultant white colonies were then
inoculated on LB-ampicillin broth.
Plasmid Isolation from the Cloned E. coli by Alkaline Lysis Method
A 1.5 mL volume of the cloned E. coli culture was centrifuged at 6500 rpm
for 7 min; the supernatant was discarded and 100 L of ice cold solution-1
(50 mM Glucose,10Mm EDTA, 25 mM Tris-Cl pH 8, Lysozyme 4 mg/mL) was
added. This was properly vortexed and 3 L RNase was added at room temperature for 5 min. A 200L of freshly prepared solution of 0.2(N) NaOH and
1% SDS was added and mixed by inverting 23 times then incubated on ice
for 5 min. A 150 L of ice cold solution-3 (60 mL 5M K- acetate, 11.5 mL
Glacial acetic acid and 28.5 mL of water) was added. The tubes were capped
and inverted 2 times then incubated on ice for 3 min (a white precipitate arose),
then it was centrifuged at 10,500 rpm for 10 min and the supernatant was
transferred into a fresh tube. Equal volume of ispropanol was added and mixed
by vortexing then it was allowed to stand on ice for 1 h. It was then centrifuged
at 10,500 rpm for 10 min, the supernatant was drained out and the tubes were
kept in inverted position. A 1 mL volume of 80% ethanol was added and tilted
up and down for 15 min, then centrifuged for 10 min at 10,500 rpm. The supernatant was drained out and the tubes were kept in open condition to evaporate
away the ethanol. This was later resuspended in 20 L of elution buffer.
Restriction Digestion
Restriction analysis of the cloned E. coli plasmid was done and the restriction mixture comprised 3 L isolated plasmid, 1 L of 10X buffer O, 0.1 L of
Sal 1, and 5.9 L of nuclease free water. The mixture was incubated in a water
bath at 37 C for 3 h. Sal 1 was used for 13 strains while EcoR 1 was used for
the rest of the strains. The restriction cut site was then checked by running on
1% agarose gel stained with ethidium bromide 0.5 g/mL.
Sequencing
Stabbed culture of the cloned was prepared and sent for sequencing at a
commercial facility known as Chromas Biotech Laboratory (Bengaluru, India).
The obtained nucleotide sequence was compared with those in the NCBI
GenBank (http://www.ncbi.nlm.nih.gov/) using BLAST (Altschul et al., 1990).

43

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I. C. Oladipo et al.

RESULTS
A total of 144 strains of the genus Enterococcus were isolated from selected traditional fermented vegetable condiments and West African soft cheese (wara).
The Enterococci strains were differentiated from other lactic acid bacteria
on the basis of their morphological appearances as well as physiological and
biochemical tests. The presumptive Enterococci strains were gram-positive,
catalase negative, oxidase negative, nonspore forming cocci in singles and pairs
with ability to grow in the presence of 6.5% NaCl at pH 9.6 and at 10 and
45 C; were able to hydrolyze esculin, pyrrolidonyl--naphthylamide, arginine;
and were unable to hydrolyze starch (Table 1). The Enterococcus strains were
differentiated based on their sugar fermentation pattern.
All the strains were able to ferment cellobiose, D-fructose, galactose, Dglucose, glycerol, lactose, maltose, mannitol, D-mannose, ribose, sucrose, and
trehalose. Enterococcus faecalis strains were able to ferment sorbitol but were
unable to ferment L- arabinose, adonitol, D-xylose, inulin, melibiose, rhamnose,
-galactisidase and -glucoronidase while E. faecium, E. casseliflavus and E.
gallinarum were able to ferment L-arabinose and melibiose. Enterococcus faecalis was further differentiated from E. faecium by the tellurite tolerance
Table 1: Morphological and Biochemical characterization of Enterococcus species.

Test Characteristics

Growth at:
4 C
10 C
45 C
pH 9.6
Growth in:
6.5% NaCl
Survival at 60 C for:
15 min
30 min
Gelatin liquefaction
H2 S production
Alpha hemolysis
Beta hemolysis
No hemolysis
Lancefield group D
Motility
Voges-Proskauer
Yellow pigment
Esculin Hydrolysis
Pyrrolidonyl
aminopeptidase

E. faecalis

E. faecium

E. casselliflavus

E. gallinarum

n = 11

n = 21

n=4

n = 108

11/0
11/0
11/0
11/0

0/21
21/0
21/0
21/0

0/4
4/0
4/0
4/0

0/108
108/0
108/0
108/0

11/0

21/0

4/0

108/0

11/0
11/0
0/11
0/11
0/11
0/11
11/0

21/0
21/0
0/21
0/21
0/21
0/21
21/0

4/0
4/0
0/4
0/4
0/4
0/4
4/0

108/0
108/0
0/108
0/108
1/107
0/108
107/1

11/0
0/11
11/0
0/11
11/0
11/0

21/0
0/21
21/0
0/21
21/0
21/0

4/0
4/0
4/0
4/0
4/0
4/0

108/0
108/0
0/108
0/108
108/0
108/0

All isolates were Gram positive cocci in singles, pairs and chains, n = No. of strains.

Genomic Characterization of Enterococcus Species

test, where E. faecalis was positive and E. faecium negative. Enterococcus

casseliflavus and E. gallinarum were motile but were differentiated by the pigmentation test. E. casseliflavus was positive for yellow pigmentation and E.
gallinarum was negative. The species were divided into four groups: E. faecalis,
E. faecium, E. cassliflavus, and E. gallinarum (Table 2).
To further ascertain the result obtained with the phenotypic methods,
30 representative strains were selected out of the 144 strains. The 30 isolates
were further characterized genotypically by the amplification of the 16S rRNA
genes. The purified DNA of the selected strains was separated on 1% agarose
gel as shown in Figs. 1ac. The molecular weight of the purified PCR product was observed to be 1500 base pairs. The purified DNA was cloned into
competent E. coli (JM 107) and plasmid isolated produced between five and
seven bands. The restriction analysis was carried out on the plasmid isolated
from the clone to ascertain the insertion. The expected restriction cut size of
Table 2: Carbohydrate fermentation pattern of Enterococcus species.
E. faecalis E. faecium E. casselliflavus E. gallinarum
Test characteristics

n = 11

n = 21

n=4

n = 108

Metabolism with:
Adonitol
L-Arabinose
Cellobiose
Dulcitol
D-Fructose
Galactose
D-Glucose
Glycerol
Inulin
Lactose
Maltose
Mannitol
D-Mannose
Melibiose
Methyl--D-glucopyranoside
Methyl--D-mannopyranoside
Methyl xylose
D-Raffinose
Rhamnose
Ribose
Sorbitol
Sorbose
Sucrose
Trehalose
D-Xylose
-Galactosidase
-Glucoronidase
Growth in 0.1% Methylene blue
Growth in 0.04% Tellurite

0/11
0/11
11/0
0/11
11/0
11/0
11/0
11/0
0/11
11/0
11/0
11/0
11/0
0/11
0/11
0/11
0/11
0/11
0/11
0/11
11/0
0/11
11/0
11/0
0/11
0/11
0/11
11/0
11/0

0/21
21/0
21/0
0/21
21/0
21/0
21/0
21/0
0/21
21/0
21/0
21/0
21/0
21/0
0/21
0/21
0/21
0/21
0/21
0/21
0/21
0/21
21/0
21/0
0/21
21/0
0/21
21/0
0/21

0/4
4/0
4/0
0/4
4/0
4/0
4/0
4/0
0/4
4/0
4/0
4/0
4/0
4/0
4/0
4/0
0/4
0/4
4/0
0/4
0/4
0/4
4/0
4/0
4/0
4/0
0/4
0/4
0/4

0/108
108/0
108/0
0/108
108/0
108/0
108/0
108/0
108/0
108/0
108/0
108/0
108/0
108/0
108/0
108/0
0/108
108/0
0/108
0/108
108/0
0/108
108/0
108/0
108/0
108/0
108/0
108/0
0/108

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I. C. Oladipo et al.

Figure 1a: Agarose gel electrophoresis of PCR products from DNA of Enterococcus species
isolated from ogiri, ugba, and okpehe. Lanes 16(A), 1: Marker Gene Ruler 100 bp plus (MBI
Fermentas), PCR amplified 16S rRNA (1500bp) gene of Enterococcus species C100, C103, C110,
C117, C120, U82, U87,U810, U817, U820, T71, T77, T83, and T88 (color figure available online).

Figure 1b: Agarose gel electrophoresis of PCR products from DNA of Enterococcus species
isolated from okpehe, wara, and iru. Lanes 16(B), 1: Marker Gene Ruler 100 bp plus (MBI
Fermentas), PCR amplified 16S rRNA (1500bp) gene of Enterococcus species T92, W184, W188,
W194, W211, W214, IW10, IW19, IP23, and IP27 (color figure available online).

Figure 1c: Agarose gel electrophoresis of PCR products from DNA of Enterococcus species
isolated from iru and dawadawa. Lanes 17(C), 1: Marker Gene Ruler 100 bp plus (MBI
Fermentas), PCR amplified 16S rRNA (1500bp) gene of Enterococcus species IP34, D100, D105,
D110, D116, and D121 (color figure available online).

Genomic Characterization of Enterococcus Species

Figure 2a: Agarose gel electrophoresis of restriction analysis of the Plasmid DNA isolated from

the cloned E. coli using sal 1. Lanes 113 (A): C100, C103, C110, C117, C120, U82, U87, U810,
U817, U820, T71, T77, and Marker Gene Ruler: DNA BST E II digest (Sigma) (color figure available
online).

Figure 2b: Agarose gel electrophoresis of restriction analysis of the Plasmid DNA isolated from

the cloned E. coli using EcoR1. Lanes 120(B): Marker Gene Ruler: DNA BST E II digest (Sigma),
T77, T83, T88, T92, W184, W188, W194, W211, W214, IW10, IW19, IP23, IP27, IP34, D100, D105, D110,
D116, and D121 (color figure available online).

4500 bp, as shown in Figs. 2a and 2b, confirmed the insertion. After confirmation of the insertion, the cloned product was sequenced. Molecular sequencing
was done bi-directionally using the forward and the reverse primers. Alignment
of the sequences with their closest match from a BLAST search was then
performed. The sequence of the strains showed 99.0% (high sequence identity) similarity with to their respective type strains (E. faecium, E. faecalis, E.
casseliflavus, and E. gallinarum) in the gene bank. The sequences were then
submitted to gene bank and accession numbers were assigned to them. They
were given accession no JN645282-JN645306, JN020631, JF774410-JF774412,
and JF915769.
The frequency of occurrence of the Enterococcus species in traditional fermented vegetable condiments and wara is shown in Table 3. Akure samples
had the lowest number of isolates with 24 been the number of organisms isolated while 30 were isolated from samples from Lagos, Ibadan, Abeokuta, and
Ilorin. The most predominant species was E. gallinarum with 75%, E. faecium
with 14.48%, E. faecalis with 7.64%, and E. casseliflavus with 2.78%, as shown
in Fig. 3.

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I. C. Oladipo et al.
Table 3: Frequency of occurrence of Enterococcus species isolated from fermented vegetable condiments and wara.

Isolates

E. faecalis
E. faecium
E. casseliflavus
E. gallinarum

Lagos

Ibadan

Abeokuta

Ilorin

Akure

n = 30

n = 30

n = 30

n = 30

n = 24

4
1
25

4
3
1
22

3
2
2
23

3
10

17

1
2

21

n = no of strains, -= not detected.

Figure 3: Percentage distribution of Enterococcus species isolated from selected traditional

fermented condiments and wara (color figure available online).

DISCUSSION
Enterococci can be encountered throughout the environment from human,
animal, and food sources. In this study, a total of 144 strains of the genus
Enterococcus were isolated from selected traditional fermented vegetable
condiments (Ugba, okpehe, iru, dawadawa, and ogiri) and West African soft
cheese (wara). This is supported by the previous investigation of Giraffa et al.
(1997) and Franz et al. (1999a), who reported that Enterococci commonly occur
in large numbers in vegetables, olives, and plant materials. They are also found
as component of the natural flora of certain foods, where they exert beneficial
effects; in certain cheeses, they are significant in ripening and flavor development. Eaton and Gasson (2001) also reported that Enterococci constitute a
major component of artisanal cheeses in southern Europe and are considered
to play an important role in ripening and aroma development. Devriese et al.
(1991) reported that Enterococci are ubiquitous and can be found free living in
soil, on plants or dairy products.

Genomic Characterization of Enterococcus Species

The emergence and dissemination of vancomycin-resistant enterococci

(VRE) underscore the importance of the rapid detection of these organisms
(HICPAC, 1995). Conventional identification of some Enterococci is difficult
because no phenotypic criteria are available to unequivocally separate the
genus Enterococcus from the other genera of gram-positive cocci. A number
of tests, such as Lancefield group D antigen, growth in 6.5% NaCl broth, the
pyrolidonylarylamidase, the leucinearylamidase, and the bile esculin test, are
all valuable for the identification of Enterococci. Unfortunately, none of these
tests alone or in combinations provides a phenotype unique to the Enterococci
(Devriese et al., 1993). Furthermore, some additional tests may have to be carried out for species differentiation (Sader et al., 1995; Singer et al., 1996).
In this study the polyphasic taxonomic approach was employed for accurate
identification of the Enterococcus strains.
The isolates were subjected to different physiological tests; the strains were
Gram positive, catalase and oxidase negative nonspore forming cocci in singles,
pairs and chains. The ability of the strains to grow in alkaline environment (pH
9.6) and at 10 and 45 C, 6.5% sodium chloride and to hydrolyze esculin agreed
with the previous studies of Franzetti et al. (2004) and Ulrich and Muller
(1998).
The sugar fermentation pattern agrees to that described by Sneath et al.
(1986) and Manero and Blanch (1999). There were variations in the fermentation of sorbitol, inulin, arabinose, rhamnose, xylose, and raffinose, but all
strains fermented cellobiose, D-fructose, galactose, D-glucose, lactose, maltose,
mannitol, D-mannose, ribose, sucrose, and trehalose without production of gas;
none fermented adonitol, ducitol, and sorbose. The ability of the strains to
ferment carbohydrates such as sucrose, melibiose, and raffinose is of special
significance. It has been reported that most legumes used in the production
of condiments contain large amounts of nondigestible carbohydrates, which
may include arabinogalactan, stachyose, sucrose, and raffinose (Irvine, 1961;
Odunfa, 1983a, b). These nondigestible carbohydrates are associated with
abdominal distention and flatulence in humans (Sarkar et al., 1997b; Naczk
et al., 1997). Hydrolysis of some of these nondigestible carbohydrates by the
strains indicates reduction or removal of the carbohydrates, which is regarded
as a safe trait of bacteria associated with fermentation of legumes containing
these carbohydrates (Yousif et al., 2005).
Identification of Enterococci using traditional phenotypic differentiation
can be tedious and time-consuming as the tests often require long incubation periods before results can be interpreted (Jurkovic et al., 2006). However,
Mohammed et al. (2009) reported that the use of molecular techniques offers a
more rapid and specific alternative. Genomic characterization has been used to
discriminate different bacteria, and the technique has been successfully used
to study the diversity among different species of Enterococcus. After amplification of the 16S rRNA gene for the 30 strains, the purified DNA obtained was

49

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I. C. Oladipo et al.

sequenced and alignment of the 16S rRNA sequences with their closest match
from a BLAST search which showed 99.0% similarity (high sequence identity) to their respective type strains, corroborating the result obtained with the
phenotypic methods.
The predominant species isolated was E. gallinarum from all the selected
traditional fermented vegetable condiments and West African soft cheese
(wara). Although, E. faecalis and E. faecium are normally reported as the
predominant species isolated from food sources (Giraffa, 2003; Klein, 2003).
On the other hand, Brtkova (2010) reported that E. faecalis, E. faecium, and
E. durans are still the most important species found in food, though recent
results proved that E. casseliflavus also may be important. The predominance
of E. gallinarum may be as a result of the environment of production of the food
sources and processing or fermentation conditions. The fact that E. gallinarum
was dominant suggests that they confer some benefit to the food. The polyphasic taxonomic approach was successfully employed for accurate identification
of the Enterococcus strains.

CONCLUSION
Identification of Enterococci by phenotypic means is often challenging because
it may be difficult to differentiate among closely related species; therefore,
molecular techniques such as PCR-based methods targeting various genes
should be encouraged. The polyphasic taxonomic approach characterization
used in this study has enabled accurate characterization of the strains.
Investigation of their functional properties would determine whether the
Enterococci strains could be used as probiotic or functional starters in the production of organoleptically unique food products which may contribute to the
local cuisine and heritage of West Africa.

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